Method and apparatus for Assay of Electrochemical Properties

ABSTRACT

The presence of a select analyte in the sample is evaluated in an an electrochemical system using a conduction cell-type apparatus. A potential or current is generated between the two electrodes of the cell sufficient to bring about oxidation or reduction of the analyte or of a mediator in an analyte-detection redox system, thereby forming a chemical potential gradient of the analyte or mediator between the two electrodes After the gradient is established, the applied potential or current is discontinued and an analyte-independent signal is obtained from the relaxation of the chemical potential gradient. The analyte-independent signal is used to correct the analyte-dependent signal obtained during application of the potential or current. This correction allows an improved measurement of analyte concentration because it corrects for device-specific and test specific factors such as transport (mobility) of analyte and/or mediator, effective electrode area, and electrode spacing (and as a result, sample volume), without need for separate calibration values. The analysis can be performed using disposable test strips in a hand held meter, for example for glucose testing.

This application claims the benefit of U.S. Provisional Applications60/496,787 filed Aug. 21, 2003 and 60/529,648, filed Dec. 15, 2003, bothof which application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

This application relates to methods and apparatus for assay ofelectrochemical properties, and in particular to methods and apparatusfor the determination of analytes, for example glucose, from smallvolume samples.

Electrochemical means to quantify or detect an analyte have often beenchosen because of their simplicity, both in terms of device manufactureand in terms of ease of use. Electrochemical sensors have often been inthe form of either potentiometric or amperometric devices.Potentiometric devices measure the effects of the charges on atoms andtheir positions; examples include the chemFET (chemical field effecttransistor) and the ion-selective electrode (including pH electrodes).Amperometric devices operate on the principle of applying a potentialand measuring the resulting current, where the magnitude of the currentgenerated is usually related to the amount of analyte present;alternatively, the total charge passed over a time may be used torepresent the amount of analyte in a region of the sample. Because therange of compounds that can generate electrochemical currents is smallerthan those that carry charges, amperometric devices can often offergreater selectivity. Much effort has therefore been concentrated inamperometric sensors in fields as diverse as environmental monitoringand medicine.

A demand for ever-increasing numbers of measurements on ever-smallersamples at a lower cost has meant that amperometric sensors are reachinga natural limit. An old form of amperometric analysis was to use aconduction cell, where the movement of species from one electrode toanother through the sample was related to its concentration. Thisapproach required careful cell-to-cell calibration to correct forvariations in electrode area and separation, which were expressed as asingle cell constant for correction of the cell reading. In more recentforms of amperometric analysis, taking readings rapidly meant onlyspecies near the investigated electrode had an effect on the result.However, with present trends towards increasingly smaller samples, theeffects of reaction at one electrode are rapidly felt as undesiredinterference at another electrode, and even if this effect can beremoved (for example by use of a silver/silver chloride cathode), thesmall sample size also means the small amount of current passed will bemore difficult to measure accurately. Furthermore, the readings fromminiature, disposable devices are made uncertain because of the limitsof manufacturing tolerance. Thus, a method and apparatus for performingthe electrochemical assay in a miniature conduction cell that would beable to produce its own correction factors for manufacturing,environmental and sample variations would be useful and beneficial.

SUMMARY OF THE INVENTION

The present invention relates to a method for evaluating a sample forthe presence of a select analyte in the sample in an electrochemicalsystem using a conduction-cell type apparatus. The method comprises thesteps of

(a) introducing the sample into a space between two electrodes of aconduction cell;

(b) applying a potential or current between the two electrodessufficient to bring about oxidation or reduction of the analyte or of amediator in an analyte-detection redox system, thereby forming achemical potential gradient of the analyte or mediator between the twoelectrodes;

(c) after the gradient is established, discontinuing the appliedpotential or current and obtaining an analyte-independent signalreflecting relaxation of the chemical potential gradient;

(d) optionally applying a potential or current between the electrodesafter the analyte-independent signal is obtained;

(e) obtaining an analyte-dependent signal during the application of thepotential or current in step (b) or step (d) or both, and

(f) correcting the analyte-dependent signal obtained in step (e) usingthe analyte-independent signal obtained in step (c) to obtain acorrected analyte-dependent signal indicative of the presence of theselected analyte in the sample.

The use of the two signals, the analyte-independent signal and theanlyte-dependent signal, allows an improved measurement of analyteconcentration over the conventional usage of an analyte-dependent signalalone because the analyte-independent signal provides information aboutdevice-specific and test specific factors such as transport (mobility)of analyte and/or mediator, effective electrode area, and electrodespacing (and as a result, sample volume), without need for separatecalibration values. This means that using the method and apparatus ofthe invention auto-calibration can be achieved that improves theaccuracy and precision of the measurement without increasing the cost.

The present invention also provides an apparatus for use in practicingthe method of the invention. The apparatus comprises a housing in whichelectronics effective to generate and observe the first and secondsignals are housed. In preferred embodiments, the housing is of a sizethat can be hand-held, and has an opening for receiving a disposablesingle use test strip of the type now known for testing of bloodglucose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reactions of an exemplary analyte-detection system foranalysis of glucose.

FIG. 2 shows an embodiment of the invention.

FIG. 3 illustrates the general operation of a conductance cell.

FIG. 4 illustrates one method for performing auto-correction inaccordance with the invention.

FIG. 5 shows results of a computer simulation of the relationshipbetween ferricyanide concentration and normalized distance across anelectrochemical cell at different times after open-circuiting the cell.

FIG. 6 shows the decay of potential as a function of time for variousvalues of P_(T).

FIGS. 7A-C illustrate an embodiment of the electrochemical cell of aconduction-cell electrochemical sensor.

FIG. 8 shows a cell with side by side electrodes.

FIG. 9 shows one example relationship than can exist between P_(T) andthe measured current for different analyte concentrations.

FIG. 10 shows representative calibration curves for different values ofP_(T).

FIG. 11 illustrates one example of the type of error that may arise ifthe value of P_(T) varies.

FIG. 12 illustrates a relationship between P_(T) and the time t taken toreach a potential difference of 0.06 V.

FIGS. 13A-C illustrates scenarios for correcting an estimate due tovariations in P_(T).

FIGS. 14A-C illustrate in flow-chart form example embodiments of theinvention.

FIG. 15 illustrates a schematic for one example embodiment of theTransducer control apparatus.

FIG. 16 shows an embodiment of a transducer control apparatus.

FIG. 17 illustrates a schematic representation of the potentiometricrelaxation signal as a function of time for different values of P_(T).

FIG. 18 illustrates in schematic form one example of quantifying ameasure of P_(T) from the measured data.

FIG. 19 illustrates in schematic form examples amperometric signals fora transient system.

FIGS. 20A-C illustrate in schematic form several example amperometricsignals that may be generated by conduction-cell based biosensorsystems.

FIGS. 21A-D illustrate in schematic form several example arrangements ofsubstantially coplanar electrodes.

FIG. 22 shows an external view of an embodiment of an apparatus inaccordance with the invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “analyte” refers to a chemical or biologicalspecies that an experiment or device is intended to detect and/ormeasure.

As used herein, the term “interferent” refers to a chemical orbiological species that is an interfering agent in the analysis of theanalyte, that is present in the sample and that contributes error to thedetection or measurement.

As used herein, the term “autocorrection” refers to a process whereinformation obtained about an apparatus during the course of use of thatapparatus is applied to other forms of information from the apparatusobtained during the course of use of that apparatus to improve theaccuracy of all or parts of the information.

As used herein, the term “conduction cell” refers to a device comprisingtwo electrodes in contact with a solution, such that the conductance ofthe solution can be calculated by passing current between theelectrodes.

As used herein, the term “conductivity cell” refers to a conductioncell.

As used herein, the term “sample factors” refers to properties and/orfactors relating to the sample solution from which an electrochemicalsignal is recorded to measure certain properties of that samplesolution. Examples include, but are not limited to, specific analyteconcentration, interferent concentrations, viscosity of the solution,dielectric of the sample, and particulate loading of the sample.

As used herein, the term “apparatus factors” refers to properties and/orfactors relating to the apparatus used to measure the electrochemicalsignal relating to the sample solution. Examples include, but are notlimited to, electrode geometry, electrode dimensions, and protectivelayers in the apparatus, which could include polymer meshes or coatings.

As used herein, the term “potentiometric relaxation” refers to change inpotential with time. An example of potentiometric relaxation includesthe change in potential between two electrodes when an applied potentialhas been removed and substantially zero current flows between theelectrodes. This change in potential can be a result of changes in theconcentration profiles of a reduced chemical species and an oxidizedchemical species in a sample with which the two electrodes are inelectrolytic contact.

As used herein, the term “environmental factors” refers to propertiesand/or factors other than “sample factors” or “apparatus factors”.Examples include, but are not limited to, temperature, humidity,physical vibrations, and ambient radio waves.

As used herein, the term “effective electrode area” refers to theelectrode area that is in electrolytic contact with the sample. Theeffective electrode area may be varied by altering the geometry of theelectrode or by partial contact of the electrode to the sample.

As used herein, the term “electrolytic contact” refers to having anelectrochemical system comprised of at least one electrode deployed in amanner so as to gather electrochemical information from a sample.Examples include, but are not limited to, an electrode in physicalcontact with a sample; an electrode separated from a sample by amembrane, a film, or other material; and an electrode separated from asample by an aqueous medium. Examples of electrochemical informationinclude Faradaic current, nonfaradaic current, and chemical potential.

As used herein, the term “steady state” refers to a condition in whichsome specified characteristic of a condition, such as but not limited toa value, rate, periodicity, or amplitude, exhibits only negligiblechange over an arbitrarily long period of time. This phrase alsoincludes a condition that exists after all or nearly all initialtransients or fluctuating conditions have damped out, and all currents,voltages, or fields remain substantially constant, or oscillateuniformly or substantially uniformly. This phrase also includesconditions that have nearly reached steady state. The term“steady-state” refers to “steady state”.

As used herein, the term “RAC” refers to redox-active compound. Theseare substances that can participate in oxidation-reduction reactions.Examples of RACs include ferricyanide, ferrocyanide, ferrocene, oxygen,and hydrogen peroxide. It will be appreciated that the identification ofa species as a redox active compound is dependent on the electrochemicalcell, and the potential differences in the cell, such that a givencompound may be an RAC in one use and non-redox active in some otherenvironment.

As used herein, the term “NRAC” refers to substances that are not RACs.

The term “mediator in an analyte-detection redox system” refers to anelectrochemical signal source which is not itself an analyte but whichis an RAC. The “analyte-detection redox system” is a system that permitsthe electrochemical detection of an NRAC-analyte. By way of example, ananalyte-detection redox system for the detection of glucose comprises anenzyme such as glucose oxidase that is capable of oxidizing glucose, andan RAC-mediator that is capable of reoxidizing enzyme to restore is toactive form. FIG. 1 shows the reaction involved in an analyte-detectionredox system for the detection of glucose.

As used herein, the term “stimulus waveform” refers to a voltage orcurrent that is applied to the electrochemical sensor system, which canbe time-varying, not time-varying, AC, and/or DC.

As used herein, the phrase “evaluating a sample for the presence of aselect analyte” encompasses both qualitative detection of the presenceof the analyte, that is whether or not the analyte is present indetectable amounts in the sample, semiquantitative detection, that iswhether or not the analyte is present in an amount greater than apredetermined threshold value, and quantitative evaluation, that isdetermination of the actual numerical amount of the analyte that ispresent.

The term “analyte-dependent signal” refers to an observedelectrochemical signal, which may be in the form of a current orchanging potential the magnitude of which is dependent on the presenceor amount of the analyte. An analyte-dependent signal need not be solelydependent on the presence or amount of analyte, and indeed theuncorrected signal discussed in this application is generally dependenton other factors besides the presence and/or amount of analyte.

The term “analyte-independent signal” refers to a signal whosetime-domain characteristics are dependent on factors other than theamount of analyte. It will be appreciated that the existence of theanalyte-independent signal is dependent on the presence of analyte, butthat the rate of decay of the signal, i.e. the time-domaincharacteristics, do not depend on analyte concentration, at least overthe range of concentrations encountered in ordinary measurements.

Method of the Invention

In accordance with a first aspect of the invention, there is provided amethod of evaluating a sample for the presence of a select analyte. Themethod includes the step of applying to the electrochemical system apotential or current sufficient to bring about oxidation or reduction ofthe analyte, or of a mediator in an analyte-detection redox system. Thisapplication of potential or current results in the formation of achemical potential gradient of the analyte or mediator across the spacebetween the two electrodes.

After the gradient is established, the applied potential or current isdiscontinued, leaving a device in which analyte or mediator in reducedand oxidized forms is distributed in concentration gradients between theelectrodes. This gradient establishes a potential difference between theelectrodes, and in the absence of an applied potential or current, thegradients and the associated potential difference relax to anequilibrium state of even distribution. The time course of thisrelaxation can be monitored by monitoring the potential difference. Thetime course of the relaxation is dependent on factors such as effectiveelectrode area, temperature, electrode spacing, hematocrit, but issubstantially independent of the concentration of analyte.

After the potential decay is monitored, an external potential isoptionally again applied to the system. An uncorrected analyte-dependentsignal is obtained from monitoring the system during either the firstapplication of potential, the second application of potential or both.Additional cycles of potential on and off could be used, and themeasurements could be made at any one or any combination of thesecycles. The uncorrected analyte-dependent signal is generally a signalthat is capable of providing, on its own, an indication of the presenceof the analyte. In preferred embodiments of the invention, the potentialis applied and the analyte-dependent signal is a current signal derivedfrom amperometric evaluation of the analyte. The analyte-dependentsignal may also be a potential difference that results from maintaininga desired current between the electrodes. Thus, the analyte-dependentsignal comprises a signal component that is dependent on thepresence/concentration of the select analyte, and may also comprisecomponents that depend on other factors, including sample factors,environmental factors and apparatus factors that are not dependent onthe presence or concentration of the selected analyte.

To provide a more accurate assessment of the analyte, it is desirable tocorrect the original analyte-dependent signal for theseanalyte-independent factors, and that is the purpose of the presentinvention. Thus, the final step of the invention is the correction ofthe uncorrected analyte-dependent signal based on the observedanalyte-independent potential decay to form a correctedanalyte-dependent signal. This signal is then preferably converted intoa user-friendly output, for example in the form of a visible displayindicating the presence or concentration of analyte in the sample.

The potential applied to the system to generate the gradient and in theoptional post-gradient-relaxation potential application step may be atime-invariant or a time-varying potential. PCT Publications WO03/060154 and WO 03/069304, which are incorporated herein by reference,each describe the usage of time-varying potentials to generate ananalyte-dependent signal.

FIG. 2 illustrates an embodiment of the invention, in which correctionis made for measurement variations that may arise in a conductance-cellelectrochemical sensor from sources other than analyte concentration.All arrows represent a set of communication channels, unless otherwiselabeled, and can include but are not limited to, electrical transmissionvia physical conductors, wireless transmission, and multiple channels ofcommunication.

As shown in FIG. 2, a transducer control apparatus (TCA) 5 applies astimulus waveform signal 10 to an electrochemical cell 50. Theelectrochemical cell 50 is based on a conduction cell and is comprisedof at least two electrodes, indicated by electrode A 55 and electrode B60. The sample 70 is in electrolytic contact with at least electrode A55 and electrode B 60 and is comprised of redox active compounds 65 andnon-redox active compounds 80. The TCA 5 has means of potentiostat andgalvanostat operation and can switch between the two modes as needed. Inpotentiostatic operation, a potential is applied and a current isgenerated. The potential is determined based on the redox properties ofthe analyte or mediator to be oxidized/reduced at the electrodes. Ingalvanostat operation, a current is applied and a potential isgenerated.

The stimulus waveform signal 10 causes an electrochemical signal 75 tobe generated at each of the electrodes, indicated by electrode A 55 andelectrode B 60 by at least one of the RACs 65 and/or at least one of theNRACs 80 in the sample 70. The signal 10 may be a current signal or apotential signal. The signal 10 may be substantially zero amps,substantially not zero amps, substantially zero volts, and/orsubstantially not zero volts. This response signal 75 is detected andmeasured by the transducer control apparatus 5. The signal 75 may be acurrent signal or a potential signal.

To facilitate digital processing of the stimulus waveform signal 10 andthe response signal 75, an analog-to-digital converter (ADC) 15 may beused to convert analog signals into a digital signal. An anti-aliasingfilter may be used in conjunction with the ADC to filter and the signalis filtered before digitizing. One of ordinary skill in the art willrecognize the possibility that such a filter may be part of the ADCitself.

A computing apparatus 25 receives the digitized signal from the ADC 15for processing. The computing apparatus is programmed to execute acorrection process 30, and includes data storage 35, for example in theform of a data storage disk, optical disk, or writable memory which canstore both program commands, reference data and results.

The correction process 30 uses functions and/or equations stored in thedata storage 35 to modify signal to correct for variations in the signalthat may arise from sources other than analyte concentration and computeuseful derived quantities. One example of a useful derived quantity isthe concentration of the desired analyte in the sample. The correctionprocess 30 can also make use of calibration data that may be containedin data storage 35.

The derived quantities are then sent to an output 45 in a useful manner.Examples of a useful manner of output are having the concentration ofthe analyte displayed to the user in a visual display or having theconcentration of the analyte transmitted and stored by electronic means.In the case of a purely qualitative determination, the useful output canbe in the form of a binary display such as a Yes/No, red/green on/offcondition in a lighted display, or audible signal.

In one embodiment of the invention, a potential is applied between theelectrodes of the cell, and current generated as a result of thisapplied potential is measured as the analyte-dependent signal. Thepotential can be applied until a steady state current is reached, andthe current is then measured. Alternatively, the current can be measuredon a current transient, before steady state is reached.

When the measurement is being taken in the method of the invention, asample is located between two electrodes and comprises anelectrochemically active species in both oxidized and reduced forms. Oneof these forms is at a concentration related to the amount of an analyteof interest. The other form is in excess. A potential difference isapplied between the electrodes that causes oxidation of the reduced format one electrode and reduction of the oxidized form at the other. Thisgenerates

(i) a difference in chemical potentials in the solution environmentsnear the two electrodes; and

(ii) an electric current in the circuitry that connects the twoelectrodes. The difference in chemical potentials creates aconcentration gradient of both forms of the electrochemically activespecies that encourages diffusion. By maintaining a steady difference inchemical potentials, the diffusion can reach a steady state and theelectric current can reach a steady level.

Removing electrical communication between the two electrodes preventsmaintenance of the concentration gradients, which begins to weakenbecause of diffusion. The weakening of the concentration gradientsresults in changes in the chemical potentials near each electrode. Thechanges can be monitored by measuring the potential difference betweenthe two electrodes. The magnitude of the measured electric current thatflowed between the two electrodes on application of a steady potentialis found to be substantially dependent on the analyte concentration andsubstantially dependent on the mobility of the electrochemically activespecies. The changes in potential between the electrodes uponelectrically isolating them are found to be substantially dependent onthe mobility, but not substantially dependent on the analyteconcentration. A measure of analyte concentration that is substantiallyindependent of mobility can be derived by suitable combination of thesetwo.

A suitable chemical potential difference can be generated by applicationof a steady potential or a potential that may vary without substantiallydestabilizing the chemical potential, either to maintain a stablecurrent (e.g. chronopotentiometry) or to stimulate other aspects of thesystem (e.g. ac impedance spectroscopy.)

An adequate perturbation of chemical potentials may be generated byapplication of an electric potential so that the change in chemicalpotentials may be monitored by subsequent electrical isolation of theelectrodes, without ever reaching a steady state in the electriccurrent. In this case, a transient current is measured that issubstantially dependent on analyte concentration and the mobility of theelectrochemically active species. The changes in potential between theelectrodes upon electrically isolating them are again found to besubstantially dependent on the mobility, but not substantially dependenton the analyte concentration. A measure of analyte concentration that issubstantially independent of mobility can therefore again be derived bysuitable combination of these two. Whilst the form of theinterdependence may vary from the steady state case, the ability toremove the mobility dependence is maintained.

In another embodiment of the invention, a current is applied between theelectrodes of the cell, and potential difference generated as a resultof this applied potential is measured as the analyte-dependent signal.The current can be applied until a steady state potential is reached,and the potential difference is then measured. Alternatively, thepotential difference can be measured on a potential transient, beforesteady state is reached.

In these example embodiments, the electrochemical cell design, stimuluswaveforms, and signal analysis processes are designed to improve themeasurement performed by a conductance-cell sensor system to reduceerrors from variations other than the concentration of the desiredanalyte.

The use of a conductance cell to measure concentration and transportproperties of chemical substances had been previously described(MacInnes, 1939). In the particular case of a charged species, such asan ion, there are typically four factors that contribute to thetransport properties of the species (Crow, 1998): concentrationgradients, potential gradients, temperature gradients, convection (e.g.by mechanical stirring). For the case of an electrochemical sensorsystem, it is generally assumed that concentration gradients andpotential gradients are the factors that contribute significantly to thetransport properties. Furthermore, Schmidt-Weinmar (1967) indicates thatconvection effects can be effectively eliminated from conductance-cellsystems by placing the electrodes less than 200 microns apart, andpreferably less than 150 microns apart.

FIG. 3 illustrates one general principle of a conventional conductancecell. The figure references a specific system employing aferricyanide/ferrocyanide redox couple. This couple is referenced as anon-limiting example, and is not intended as an indication that thiscouple is the only one that can be employed. Two electrodes 100 and 105are placed in a substantially parallel configuration in electrolyticcontact with a sample containing the species of interest. The geometryof the conduction cell does not limit the invention. The principles ofoperation are valid with many other geometries, including non-parallelfacing configurations, different areas for each electrode, and coplanarconfigurations. In this example, the redox active compounds (RACs) areferricyanide 125 and ferrocyanide 120, which form a redox couple. Apotential source 110 imposes a potential difference between the twoelectrodes. In this example, Cathode Electrode 100 acts as the cathode,where a reduction reaction occurs to convert ferricyanide 125 toferrocyanide 120; and Anode Electrode 105 acts as the anode where anoxidation reaction occurs to convert ferrocyanide 120 to ferricyanide125. In this process, an electron 115 is transferred from the anode 105to the cathode 100 for each molecule that reacts at a given electrode.Arrows 135 and 130 represent a transport process, such as diffusion,which contributes to the transport of species in the sample.

In some embodiments of the invention, the measured analyte species maybe produced, consumed, and/or altered by other chemical reactions. FIG.1 illustrates one example of this in an enzyme-linked biosensor where asubstrate, such as glucose 500, reacts with an enzyme, such as glucoseoxidase, to convert the enzyme from an oxidized state, GODox 510 to areduced state, GODred 515. Ferricyanide 525, for example, can react withthe reduced enzyme GODred 515 to convert it to its oxidized form GODox510, in the process being reduced to ferrocyanide 520. Thus, the amountof ferricyanide and ferrocyanide, in this example, may be changed byother processes which may occur in the sample either before, during, orafter measurement. The determination of ferrocyanide concentration, inthis example, may then be related to the concentration of glucose.

Conduction cells such as the one illustrated in FIG. 3 can be used fordetermining the concentration in and transport properties of the analyteor RAC species through the sample medium (MacInnes, 1939). Such cellscan be used by applying either a DC potential or an AC potential betweenthe electrodes. AC potentials have been used to minimize theelectrochemical reaction products at each electrode; however, dependingon the needs of the application, either method could be used todetermine transport properties of the analyte.

To determine the transport properties of a conductance cell, the currentthat flows through the cell in response to a voltage is measured. Theresistance of the cell is computed by taking the ratio of the appliedvoltage to the resulting current. The conductivity, κ, of the sample maybe computed by:

$\kappa = \frac{h}{RA}$

where h is the distance between the electrodes, R is the resistance, andA is area of each electrode, which are assumed to be equal. The cellconstant of a conductance cell, K_(cell), is defined by the quantity:

$K_{cell} = \frac{h}{A}$

Thus, if the cell constant for a conductance cell is known, then theconductivity of an unknown sample may be determined by measuring theresistance across the cell as follows:

$\kappa = \frac{K_{cell}}{R}$

Since the conductivity of the sample is a function of the concentrationand transport properties of the analyte, the prior art has thusestablished a method of determining transport properties such asdiffusion coefficient and mobility of an analyte due to concentrationgradients and potential gradients, respectively, that can exist in aconductance-cell electrochemical sensor. How conductivity relates tofactors such as transport properties and concentration depends on thespecific nature of the experimental set up.

For this method to be successful, the cell constant must be known. Theconventional method for doing this is to calibrate the cell using asample of known conductivity to determine the cell constant and then usethe same cell to measure the conductivity of an unknown sample(MacInnes, 1939).

Variations on this method are also known. Conductance measurements havebeen routinely used to determine both concentrations and transportproperties in samples. For example, in water purification, theconcentration of ionic species has been determined by conductancemeasurements. In another example, the diffusion properties of an analytehave been used to determine the level of particulate matter in a sample.In particular, one can use conductance measurements for determining thelevel of hematocrit in blood samples. One example of when this becomesparticularly important is for electrochemical blood glucose sensors; thesensor reading may be significantly affected by the level of hematocritor the viscosity of the blood sample since the transport properties,such as diffusion and/or migration, are affected.

For example, particulate matter such as hematocrit, and other factors,such as protein content, chylomicrons, and platelets, can affect thetransport properties of many of the chemical species involved inperforming blood-glucose measurements. Thus, much interest has beenshown in quantifying the effects that transport properties have on thedetermination of analyte concentrations, such as glucose, that arecomputed by analytical instruments. Many factors can affect thetransport, including but not limited to migration due to an electricfield, diffusion due to a concentration gradient, and convection due tomovement of the sample or temperature; and that an analogous approachmay be used to correct for transport variations resulting from thesefactors. Convection effects can be minimized—and effectively removed—byhaving the electrodes in a conduction-cell system spaced less than 200microns apart, and more preferably, less than 150 microns apart(Schmidt-Weinmar, 1967).

FIG. 3 illustrates one example embodiment of a cell that can be used formeasurement. In this case, the target analyte is ferrocyanide 120, whichin this example is taken to be a minority species. Ferricyanide 130 inthis example is taken to be in excess, thereby being a majority species.Applying a sufficiently large electric potential between the electrodes100 and 105 in contact with the sample will change the chemicalpotential of the nearby solution as the concentration of the minorityspecies at one of them falls very close to zero while its concentrationat the other electrode approximately doubles. In one example, ifElectrode A 105 is at a sufficiently higher potential than Electrode C,then an oxidation process occurs at electrode A 105 and a reductionprocess occurs at Electrode C 100. One of ordinary skill in the art willrecognize that the applied potential can also affect the distribution ofionic species and that this can be more fully expressed by theelectrochemical potential, which includes the effects of this. Theunderlying pattern of behavior discussed here in terms of the chemicalpotential is therefore for illustrative purposes only and not to betaken as a convention. Other expressions of forces of equilibration onthe species than those specifically disclosed herein are within thescope of the present invention. Examples of such forces include athermodynamic force such as from a concentration gradient described inAtkins (1999) and force due to motion of the medium as in convection.

In an example of a method to extract the cell constant, K_(cell), byapplying a voltage to the cell and then removing electricalcommunication, drawn from an equivalent circuit model, the amount ofcharge stored in the conduction cell by creating concentration gradientscan be expressed as:

Stored charge=nF*[ferrocyanide]*(Volume of cell)

A capacitance is therefore produced by charging to the applied voltageVapp.

Capacitance=(stored charge)/Vapp

Capacitance=nF*[ferrocyanide]*(Volume of cell)/Vapp

Removing the electrical communication between the electrodes leaves thestored charge to discharge itself exclusively through the resistance Rof the cell. In this equivalent circuit model, the time constant of thisdischarge can then be determined by standard methods from the variationof potential between electrodes over time, where:

Time constant=R*Capacitance

Since conductivity, κ=Kcell/R=γ*[ferrocyanide]where γ is a constant of proportionality, this implies

R=Kcell/(γ*[ferrocyanide])

hence, the

time constant=nF*(Volume of cell)*Kcell/(γ*Vapp)

which is a concentration independent measure of Kcell.

Another example embodiment from the perspective of ion mobilitiesconsiders a potential gradient along the distance between the twoelectrodes that will be created by applying a voltage to the cell.Movement of species along this gradient has been described in the priorart for current in a conduction cell (MacInnes, 1939). The movement offerrocyanide in such as system has been described in terms of itsmobility, U, in response to an electric field, {right arrow over (E)}.This equation appears as:

$I_{s} = {\beta \; \overset{->}{E}U\; A\; \frac{C}{h}}$$K_{cell} = \frac{h}{A}$ $s = {{- U}\; \overset{->}{E}}$$I_{s} = {{- \beta}\; C\; A\; \frac{s}{h}}$

where I_(S) is the steady-state current, β is a proportionalityconstant, s is the drift speed and {right arrow over (E)} is theelectric field. The ratio of h to A was typically calculated in acalibration step using a known standard to give the cell constant,K_(cell). (Atkins, 1999). The drift speed is given as a negative numberin this example since the current is taken to be carried by negativeions; thus, the drift speed of negatively charged ions will be in theopposite direction to the applied electric field. Other equations arepossible for I_(S), depending on the nature of the apparatus. In thisexample, there are a number of variables that can distort extractedconcentration information from the electrochemical current. In practicalmeasurements, the cell constant can vary from cell to cell sincemanufacturing variations can alter the geometric factors affecting thecell constant. Furthermore, when analyzing real samples, such as wholeblood for glucose determination, the transport properties of theanalytes—such as drift speed—may well vary between samples. For example,the level of hematocrit is known to affect the movement of chemicalspecies in blood. Thus, signal variations due to factors other than theconcentration, such as the variations of the cell constant and the driftspeed, can significantly alter the estimated glucose concentration froman electrochemical sensor system.

The difficulty, therefore, is being able to quantify the errorintroduced by variations other than the analyte concentration, such asvariations in the cell geometry and in the transport properties, tocorrect for estimates of analyte concentration. A method and apparatusfor performing such an autocorrection would be useful. Embodiments ofthis invention describe a novel apparatus and method for determining andcorrecting for the environmental sources of measurement variation—thatis, sources other than concentration—without a priori knowledge of theelectrode area, the electrode separation, drift speed, or the mobility.

An analogous method and apparatus may be used for the motion of speciesdue to a concentration gradient. In this example embodiment, aconcentration gradient along the distance between the two electrodeswill be created by the electrochemical reactions and movement of speciesalong this gradient may be described by Fick's law of diffusion:

${Flux} = {{- D}\frac{d\; c}{d\; x}}$

where D is a diffusion coefficient, dc/dx is the concentration gradientalong a particular axis and Flux is the amount of substance that movesthrough a unit area perpendicular to this axis in a unit time. In oneexample where the flux reaches a steady state and where the electrodesare parallel, planar structures with the sample between them giving awetted area, A, on each electrode and where the electrodes are separatedby a distance, h, then the flux for an initial minority carrierconcentration, C, will be:

${Flux} = {{- D}\; \frac{2C}{h}}$

If n electrons are exchanged per molecule of minority carrier consumedelectrochemically at one electrode or generated electrochemically at theother, then the steady-state current, I_(S), generated by the movementof minority carrier may be given by:

$I_{s} = {n\; F\; D\; A\; \frac{2C}{h}}$

where F is Faraday's constant. Thus, the movement of an ion in terms ofits mobility U under the force of an electric field {right arrow over(E)} is equivalent to its movement in terms of its diffusion coefficient(with suitable adjustment of units) and so the term nFD can be expressedas β{right arrow over (E)} U (Atkins, 1999). Thus, the invention may beapplied to a system in terms of its diffusion as well.

According to an embodiment of the invention, the estimate of analyteconcentration may be improved by determining and correcting for theerrors introduced by unknown variations in apparatus factors, such asthe cell geometry, and sample factors, such as the sample composition. Asignificant challenge in chemical sensors is to determine theconcentration of the target analyte accurately since variations in themeasurement environment—such as electrode separation or viscosity—canaffect the estimate of analyte concentration. An apparatus and methodthat is sensitive to the effects of such variations but is substantiallyindependent of analyte concentration could be used for estimating andcorrecting for these sources of variation.

According to an embodiment, the error in estimating analyteconcentration that arises from different effective electrode areas canbe reduced by using an apparatus and method that is sensitive to theeffects of varying electrode areas but is independent of, or lessdependent on, analyte concentration. This could be used for determiningelectrode area and thus allowing a correction to be applied to theconcentration estimate that accounts for this variation.

According to an embodiment, the error in estimating analyteconcentration that arises from different transport properties ofanalytes in samples can be reduced using an apparatus and method that issensitive to these properties but is independent of, or less dependenton, analyte concentration. This could be used to determine themobilities of analytes in samples and thus enable a correction to beapplied to the estimate of analyte concentration that corrects forthese.

Embodiments of the invention relate to determining certain parameters ofthe measurement system that can affect the measured signal and thusaffect the estimated concentration. In one embodiment, the geometriccell parameters and the transport properties of the sample are taken asthe representative sample factors and apparatus factors—other thananalyte concentration itself—which can affect the measured signal. Theeffects of one component of the cell constant, h, and drift speed, s,may be combined into an Effective Transport Parameter (P_(T)) and may beconsidered separately from the electrode area term. In particular P_(T)may be given by:

$P_{T} = {- \frac{h}{s}}$$C = {{- \frac{K_{cell}I_{s}}{\beta \; s}} = {{- \frac{I_{s}h}{\beta \; s\; A}} = \frac{I_{s}P_{T}}{\beta \; A}}}$

One of ordinary skill in the art will recognize that an analogousexpression may be given for a system under the influence of aconcentration gradient which is better described in terms of a diffusioncoefficient. In this case, P_(T) can be given as follows:

$P_{T} = \frac{h}{D}$$C = {\frac{\alpha \; K_{cell}I_{s}}{D} = {\frac{I_{s}h}{2n\; F\; D\; A} = {\frac{I_{s}P_{T}}{2\; n\; F\; A} = \frac{\alpha \; I_{s}P_{T}}{A}}}}$$\alpha = \frac{1}{2\; n\; F}$

The effective electrode area, A, can be found by a method as describedin our pending patent application number WO 03/069304. Thus, a method todetermine P_(T) allows for complete characterization of the measurementconditions and enable a more accurate estimate of the concentration. Ina preferred embodiment, P_(T) is determined substantially independent ofelectrode area and substantially independent of analyte concentration.However, this is not a requirement and does not limit the scope of theinvention. An embodiment of the invention describes a method andapparatus that enable both the apparatus factors—such as the geometricproperties of the cell—and sample factors—such as transport propertiesof the sample—to be found simultaneously, allowing for more completeautocorrection for variations in the measured signal that arise fromenvironmental factors, sample factors, and apparatus factors. Prior artin the use of conduction cells has typically required that eachconduction cell be calibrated in a separate step such and does notprovide a means for auto-correcting for variations in the measuredsignal that arise from environmental factors, sample factors, andapparatus factors.

FIG. 4 illustrates one method for performing auto-correction of suchvariations to increase the accuracy of the estimated analyteconcentration. This diagram is an illustrative embodiment and does notlimit the invention. One of ordinary skill in the art will recognize thepossibility that these steps do not necessarily have to be executed inthe stated order. A sample is applied to the cell (step 200), then apotential signal is applied to the cell (step 205). This potentialsignal should be such that a reduction and/or oxidation process occursat at least one of the electrodes in the cell. One of ordinary skillwill recognize that the potential signal need not necessarily be appliedafter the sample is applied to the cell but that the sample may beapplied to the cell after the signal is applied. A steady-state currentis determined from the cell (step 210). This current does notnecessarily need to be a time-invariant current since time-varyingcurrents may also be classified as steady-state if the characteristicsthat describe the signal are approaching a stable value.

Once this steady-state current has been determined, the cell is thenopen circuited (step 215) and the transient potential between the twoelectrodes is determined (step 220). One of ordinary skill in the artwill recognize the possibility of other methods to maintainsubstantially zero electronic current between the electrodes other thanby open-circuiting the cell. An example of another method includes theuse of a high-impedance switch such as a transistor. The exampleembodiment of open-circuiting the cell (step 215) is an example and doesnot limit the invention. Correction factors for the environmentalvariations, for example those arising from variations in the driftspeed, mobility, diffusion coefficient and/or from the cell constant,may be determined (step 225) based on information from the steady-statecurrent before the cell has been open circuited and from the transientpotential after the cell has been open circuited. The correction factorscan then be used to correct the measured steady-state signal forvariations caused by the environmental sources (step 230) and thecorrected concentration estimate can be computed and output in a usefulform (step 235). One of ordinary skill will recognize the possibilitythat the correction of the environmental sources of variations need notbe a separate distinct step but may be integrated with the computationof the concentration. One of ordinary skill will also recognize thatinformation of other parameters of the sample than analyte concentrationmay also be derived from the correction factors as separate, valuableinformation. Examples of such parameters include hematocrit,temperature, and viscosity.

In one embodiment, after I_(S) is determined, the circuit is opened, andthe transient potential between the electrodes is determined. Oneembodiment of realizing this is to have the measuring and controlapparatus switch from a potentiostatic operating mode, where a potentialis applied and a current is determined, to a galvanostatic operatingmode, where a set current is maintained—in this case nearly 0 Amps—and apotential is determined. One of ordinary skill in the art will recognizethat other embodiments—other than open-circuiting the cell—are possiblefor achieving substantially zero amps. One example is to use ahigh-impedance switch such as a transistor to restrict the currentflowing in the circuit to substantially zero amps. The example ofopen-circuiting the cell is an example embodiment and does not limit theinvention.

Once a steady-state current has been established, there will be aconcentration gradient between the two electrodes. A factor that isrelated to P_(T) can be determined by measuring the rate of relaxationof the electrode potentials upon removal of an imposed voltage betweenthe electrodes. In the absence of an imposed voltage, the steady-statedistribution of species—for example, such as a concentrationgradient—will be unstable and the electroactive species will move in anattempt to restore a more stable concentration profile of moleculesthroughout the sample. The different relative concentrations offerrocyanide ions and ferricyanide ions at each electrode will givedifferent chemical potentials, and these chemical potentials will changewith time as these concentrations equilibrate. This information can thusbe monitored using potentiometric methods, and the measured change inpotential with time can be related to P_(T). Examples of methods todetermine a measure of P_(T) include:

1. The time from the point of removal of the imposed voltage for thepotential to reach a particular value

2. The potential at a particular point in time after removal of theimposed voltage 3. A measure of the rate of decay of the potential afterremoval of the imposed voltage such as:

-   -   The slope of the plot of the potential vs. time during a        particular period of time    -   The slope of the plot of the logarithm of the potential vs. time        during a particular period of time    -   The slope of the plot of 1/V² vs. time during a particular        period of time, where V is the potential        Other quantities can be used to determine a measure of P_(T)        from monitoring the change in potential with time.

In the example of a conductance-cell sensor, the relaxation of theconcentration profile upon removal of the imposed potential differencecan be described by the following relationships (Atkins, 1999):

$J = {{- D}\; \frac{c}{x}}$ J = sc

If the conductance-cell sensor is switched from amperometric operationwhere a potential is applied to potentiostatic operation where theimposed potential is removed—and, in this example, a substantially zerocurrent is maintained—and the potential is measured at time t=0, then atthe initial relaxation stage (that is, at t=0+) the concentrationgradient may be given as follows, and P_(T) may be computed:

$\frac{c}{x} = \frac{2c}{h}$${sc} = {{{- D}\; \frac{c}{x}} = {{- D}\; \frac{2c}{h}}}$$s = {- \frac{2D}{h}}$$P_{T} = {\frac{- h}{s} = \frac{h^{2}}{2\; D}}$

In contrast to monitoring currents (see, e.g., U.S. patents: U.S. Pat.No. 5,942,102, U.S. Pat. No. 6,179,979, U.S. Pat. No. 6,284,125),monitoring the chemical potentials at the electrode will therefore allowfor a measurement that is independent of the area of the electrode. Whenthe steady-state concentration gradient relaxes under potentiometricconditions (i.e. no electrochemical transfer of charge from oneelectrode to another via an electronic current) the concentrationprofile changes, and a computer-simulated model of this is shown in FIG.5.

The simulation results shown in FIG. 5 model the cell illustrated inFIG. 5 where Anode Electrode 105 is located at x=0 in FIG. 5 and CathodeElectrode 100 is located at x=1 in FIG. 5. The distance between theelectrodes has been normalized to 1 unit. The concentration profile offerrocyanide can be seen to evolve with time. A steady-stateconcentration profile exists when a steady-state current is realized andbefore the circuit is opened, given by 300. The circuit is opened at t=0seconds and the concentration profile is shown at the following times:t=0.2 sec 305, t=0.4 sec 310, t=0.6 sec 315, t=0.8 sec 320, and t=1.0sec 325. The chemical potential difference between species at what wasthe anode (Anode Electrode 105) and cathode (Cathode Electrode 100) canbe described by:

$V = {{\mu_{anode} - \mu_{cathode}} = {\frac{n\; F}{RT}\left( {{\ln \left( \frac{{a({ferrocyanide})}_{anode}}{{a({ferricyanide})}_{anode}} \right)} - {\ln \left( \frac{{a({ferrocyanide})}_{cathode}}{{a({ferricyanide})}_{cathode}} \right)}} \right)}}$

where μ is the chemical potential of the ferrocyanide/ferricyanidecouple, a(species) is the activity of that species, and subscripts referto the position in the cell. The activity of the species is related tothe concentration, but is a more ideal form that accounts for deviationsin thermodynamic quantities from those predicted purely byconcentration; however, using concentrations, the potential differenceat the electrodes can be approximated by:

$V = {\frac{n\; F}{RT}\ln \; \frac{\lbrack{ferroycyanide}\rbrack_{anode}}{\lbrack{ferrocyanide}\rbrack_{cathode}}}$

where [ferrocyanide]_(electrode) is the concentration of ferrocyanide atthe appropriate electrode, and [ferricyanide] is assumed to be in largeexcess throughout the sample and so may be approximated as remainingsubstantially constant throughout the sample and at the various times ofinterest. The evolution of this voltage over time has been modeled andis presented in FIG. 6 for various values of P_(T). In this figure, thepotential is determined as it is relaxing after the circuit has beenopened at time t=0 for the following values of P_(T): P_(T)=28.7 s 420;P_(T)=19.2 s 415; P_(T)=11.6 s 410; P_(T)=5.9 s 405; P_(T)=3.8 s 400.There is a clear effect of P_(T) on the time constant of the relaxation;it will be clear to one ordinarily skilled in the art that otherrelationships may exist depending on apparatus factors, sample factors,and/or environmental factors, such as the cell configuration andmeasurement method.

Potentiometry measurements can therefore be used to determine P_(T) fromthe variation between measured potential and time; the evolution of thepotential relaxation is substantially independent of the analyteconcentration and electrode area but is a function of P_(T), therebyproviding a needed method for determining a correction factor forvariation in P_(T). It will be clear to one of ordinary skill in the artthat there are a variety of methods of using this potential variation toquantify the effect of P_(T). Examples of such methods includedetermining a slope of the potential relaxation during an interval oftime, determining the time it takes to reach a particular potentialvalue, and/or determining a time constant for the decay rate for thepotential relaxation.

FIG. 6 illustrates one example embodiment of determining a measure ofP_(T). In this example, the time taken to reach a potential differenceof 0.06 V after switching to potentiometric operation is measured fordifferent values of P_(T). It is clear that the time taken to reach agiven potential difference upon potentiometric relaxation of theconduction cell can be a measure of P_(T) and therefore, the embodimentsof the invention provide a means for determining a measure of P_(T).When a value for the effective electrode area is determined, thenvariations in sample factors, apparatus factors, and environmentalfactors (including factors such as the effects of cell geometry andtransport of analyte) which can contribute to variations in effectiveelectrode area and/or variations in P_(T) can be corrected to determinea more accurate estimate of analyte concentration. For example, onemethod of determining an effective electrode area has been disclosed inPCT 03/069304 and is also described above. This method involves theapplication of a small amplitude sine wave to the electrodes andrelating the resulting sinusoidal current to the electrode area via aset of appropriately constructed equations. When a sample is measured bysuch a conductance-cell electrochemical sensor, I_(S) can be directlymeasured and determined, P_(T) can be determined by the method andapparatus of embodiments of the invention, A can be determined by thepreviously described methods, and other constants can be computed apriori, thereby giving a more accurate estimate of the concentration.

FIGS. 7A-C illustrate an embodiment of the electrochemical cell of aconduction-cell electrochemical sensor. FIG. 7A shows a compositethree-dimensional schematic drawing for such an apparatus; FIG. 7B showsa schematic drawing of a side view; and FIG. 7C shows a schematicdrawing of some of the components that comprise the apparatus separatedfrom a composite construction. This example embodiment is comprised of aconduction cell with substantially parallel electrodes (1320 and 1325)separated by a volume 1340 which can hold a sample and substantiallydefines a sample chamber. This volume 1340 is herein referred to as“sample chamber”. Each of the electrodes (1320 and 1325) is supported bya substantially nonconductive material (1300 and 1305). Each of theelectrodes (1320 and 1325) has electrical connections provided bysubstantially conductive paths (1335 and 1330) that are also supportedby a substantially nonconductive material (1300 and 1305). The thicknessof the electrodes (1320 and 1325) may be substantially the same as,substantially less than, or substantially greater than the thickness ofthe leads (1330 and 1335). The two electrodes are kept separated bysubstantially nonconductive material (1325 and 1310). The volume wherethe sample is placed (1340) may be partially defined by the electrodes(1320 and 1325) and/or partially defined by the substantiallynonconductive material (1320 and 1315).

In an exemplary embodiment, two facing, and substantially parallel,sides of the sample chamber 1340 are substantially defined by the areaspanned by two electrodes (1320 and 1325), as illustrated in theschematic of FIGS. 7A-C. The apparatus of FIGS. 7A-C illustrates oneexample embodiment of the electrochemical cell 50 illustrated in FIG. 2.One of ordinary skill in the art will recognize that other embodimentsare possible. For example, the electrodes need not be substantiallyparallel to each other. In another example, the electrodes may lie inthe same plane, and illustrated in FIG. 8. In this example embodiment,electrodes 2005 and 2020 lie in the same plane on a substantiallynonconductive substrate 2000. Electronics connectors 2010 and 2015provide a means of electronic coupling between the electrodes (2005 and2020) and the TCA. Thus, there are many different geometricconfigurations that can be used for a conductance-cell sensor. Theexample discussed in this document is one example embodiment and doesnot limit the invention.

In the apparatus of FIGS. 7A-C, the two electrodes (1320 and 1325) canbe operated as either Electrode A 55 or Electrode B 60. The sample 70 issubstantially located in the sample chamber 1340. Electrical contactwith the Transducer Control Apparatus 5 is achieved via thesubstantially conductive paths (1335 and 1330) which can beelectronically coupled to the Transducer Control Apparatus 5 to providea substantially conductive electronic path from the Transducer ControlApparatus 5 to the electrodes (1320 and 1325). One example embodiment ofelectronically coupling the Electrochemical Cell of FIG. 13 to theTransducer Control Apparatus 5 is to provide a means of substantiallycontacting a region of each of the substantially conductive leads (1330and 1335) to a portion of the TCA 5. One exemplary example of such ameans is to substantially contact that region of each of thesubstantially conductive leads that are furthest away from the region incontact with the electrodes (1320 and 1325). In the example schematicillustration on FIG. 7A-C this is illustrated by that portion of thesubstantially conductive leads (1330 and 1335) that extends beyond thesubstantially nonconductive material 1310. One of ordinary skill in theart will recognize the possibility of other means of electronicallycoupling the Electrochemical Cell 50 illustrated in FIGS. 7A-C to thetransducer Control Apparatus 5.

In one exemplary example embodiment, the apparatus of FIGS. 7A-C can beused according to the process illustrated in FIG. 3. In one embodiment,one of the electrodes 1320 in the cell is the Cathode Electrode 100 andthe other electrode 1325 is the Anode Electrode 105. In anotherembodiment, one of the electrodes 1325 in the cell is the CathodeElectrode 100 and the other electrode 1320 is the Anode Electrode 105.The ferricyanide 125 and the ferrocyanide 120 are substantially locatedin the sample chamber 1340. The voltage source 110 is provided by theTransducer Control Apparatus 5 and the current 115 travels along asubstantially conductive path that is in part comprised of thesubstantially conductive leads (1330 and 1335). The transport processes(130 and 135) substantially occur inside the sample chamber 1340.

Apparatus of the Invention

A further aspect of the present invention provides an apparatus for usein practicing the method of the invention. Thus, the invention providesan apparatus for determining the presence of an analyte in a sampledisposed in an electrochemical cell said electrochemical cell comprisingtwo electrodes between which the sample is placed for analysis, saidapparatus comprising:

(a) a housing having a space for receiving the electrochemical cell;

(b) means for applying a potential or a current between the twoelectrodes of the electrochemical cell when it is received within thehousing (for example a potentiostat or a galvanostat);

(c) means for measuring oxidation or reduction of an analyte or amediator in an analyte-detection system occurring within theelectrochemical cell when the potential or current is being applied(e.g. a circuit for measuring/observing a current or a potentialdifference between the electrodes);

(d) means for switching the potential or current off after a period oftime during which a chemical potential gradient is established betweenthe two electrodes (e.g. a switch that open circuits the cell, or ahigh-impedance switch);

(e) means for monitoring the decay of the chemical potential gradientafter the potential or current is switched off (e.g. a circuit forobserving the potential difference between the electrodes);

(f) programmed data processing means for combining the measuredoxidation or reduction with the monitored decay to produce an indicationof the presence of the analyte in the sample (e.g. a data processor withaccompanying programming that performs the steps described in thisapplication); and

(g) output means for conveying the indication of the presence of theanalyte in the sample to a user.

The apparatus may be supplied separately, but is generally used incombination with an electrochemical cell in the form of a single usetest strip. The apparatus has a slot for receiving the test strip, andappropriate signal generating and processing elements for applying thepotential and current and for monitoring the result current or potentialand the decay of the chemical potential gradient, and for converting theresulting information into an indication of the results of theevaluation. The test strip may be any test strip appropriate for thedetection of the particular selected analyte. In preferred embodiments,the strip has facing electrodes and a spacing between the electrodesthat is sufficiently short that the gradient of oxidized and reducedspecies extends across at least 10%, more preferably more than 80%, upto 100% of the distance between the electrodes. In general, this will bea distance of 20 to 400 microns. A display may be incorporated as partof the meter, for example in the form of an LCD display, an LED display,or moving coil meter. The display may also be separate from the meter,and connected with a wired or wireless communications link to the meter.

FIG. 22 shows an external view of an embodiment of the apparatus of theinvention. Housing 3000 can be made from any suitable material, but willmost commonly be made of an impact resistant plastic. Housing 3000 hasan opening 3005 for receiving a test strip that comprises electrodes andconnectors for making electrical contact between the test strip and theapparatus. A display 3010 provides output in a form readable by theuser. Optionally, the apparatus can include a start button 3015,although detection of an inserted test strip may also be used to startthe apparatus for processing of an analyte test.

Determination of Effective Electrode Separation

Independent of or in conjunction with the determination of analyteconcentration, the methods of the invention can be used to determine theeffective electrode separation between two electrodes in anelectrochemical cell. Thus, in a further aspect, the present applicationprovides a method for determining the effective separation distancebetween a first electrode and a second electrode in an electrochemicalcell, the method comprising the steps of:

applying an external force in the form of an applied potential or anapplied current to generate a chemical potential gradient between thefirst electrode and the second electrode;

stopping the application of the external force;

observing the decay of the chemical potential gradient as a function oftime; and

computing the effective electrode separation distance from the observeddecay of the chemical potential gradient.

Determination of an Effective Transport Property

Independent of or in conjunction with the determination of analyteconcentration, the methods of the invention can be used to determine aneffective transport property of an electrochemical system. Effectivetransport properties include mobility of species, diffusioncharacteristics of species, and combinations of these properties withthe effective electrode separation.

The following examples illustrate example embodiments of the method andapparatus of the invention.

Example 1 Correcting for Variations in P_(T)

One example of using P_(T) to increase the accuracy of electrochemicalsensor measurements is discussed. FIG. 9 shows one example relationshipthan can exist between P_(T) and the measured current for differentanalyte concentrations. The data in FIG. 9 was simulated according to aconduction-cell electrochemical sensor as illustrated by the exampledrawings in FIG. 3 and FIGS. 7A-C. FIG. 9 illustrates the variation incurrent (given in amps) for different values of P_(T) for a sensorcomprised of each electrode having an effective surface area of 1 cm².Data points are shown in FIG. 9 for the steady-state current that isgenerated by the application of a voltage difference of 0.4 V betweenthe two electrodes for a sample comprised of 2 mM ferrocyanide 1400 and1 mM ferrocyanide 1405. It is clear that the measured current isdependent upon both the concentration of analyte and the value of P_(T).Thus, variations in the value of P_(T) can cause signal variations thatcan introduce error into the measurement of analyte concentration.

FIG. 10 illustrates this problem further by showing representativecalibration curves for different values of P_(T). In this figure, theconcentration is given on the x-axis in mM, and the measuredsteady-state current is given on the y-axis in amps. Data points 1500correspond to a P_(T) value of 44.3 s; data points 1505 correspond to aP_(T) value of 20.6 s; data points 1510 correspond to a P_(T) value of6.58 s. Again, the error in analyte estimation that can arise fromvariations in P_(T) is illustrated. If one particular calibration curveis taken to be the reference calibration curve, then this implicitlyassumes that a particular value of P_(T) is associated with themeasurement. However, if the value of P_(T) varies when an unknownsample is measured, then the resulting estimate in analyte concentrationmay be erroneous.

FIG. 11 illustrates one example of the type of error that may arise ifthe value of P_(T) varies. In this example, the calibration curve iscomprised of data points 1505 from FIG. 10; this calibration curve wasdetermined for a system with a value of P_(T) equal to 20.6 s. Anequation to describe this calibration curve is:

$I = {{{\left( {{4.673\; E} - 6} \right)\lbrack{ferrocyanide}\rbrack}\lbrack{ferrocyanide}\rbrack} = \frac{I}{\left( {{4.673\; E} - 6} \right)}}$

where I is the measured current, and [ferrocyanide] is the concentrationof ferrocyanide in the sample. Thus, a measurement of the current can beused to estimate the concentration of analyte—in this example,ferrocyanide—in a sample by using the above equation.

FIG. 11 illustrates the error which may occur in estimating the analyteconcentration if the value of P_(T for the meaurement) were to bedifferent from the value of P_(T) that was used when determining thecalibration curve. Current measurements are determined from a systemwith a 2 mM ferrocyanide concentration for different values of P_(T). Asthe value of P_(T) varied, the measured current varied. Since theestimation equation is determined from a calibration curve that wasdetermined from a system that has a particular value of P_(T), ifmeasurements are made with a system that has a different value of P_(T),the resulting estimation equation will yield an inaccurate estimate.FIG. 11 illustrates the error in analyte estimation that can occur fordifferent values in P_(T). The y-axis of the chart in FIG. 11 is thepercent error in the estimate of ferrocyanide concentration asdetermined by the following:

${\% \mspace{14mu} {Error}} = {{100 \times {\frac{\lbrack{ferrocyanide}\rbrack_{estimate}}{\lbrack{ferrocyanide}\rbrack_{actual}}\lbrack{ferrocyanide}\rbrack}_{estimate}} = {{\frac{I_{measured}}{\left( {{4.673E} - 6} \right)}\lbrack{ferrocyanide}\rbrack}_{actual} = {2{mM}}}}$

I_(measured) is the measured current; [ferrocyanide]_(actual) is theactual ferrocyanide concentration in the sample (2 mM, as indicatedabove); [ferrocyanide]_(estimate is) the estimated ferrocyanideconcentration as determined by an equation describing the calibrationdata 1505. FIG. 16 illustrates an example of the error in estimatinganalyte concentration that can occur if the value of P_(T) changes fromthe value that was used when determining the calibration data. The datapoints represent the percent error in estimating the analyteconcentration for different values of P_(T). For reference, it should benoted that a value of P_(T) equal to 20.6 s was used when determiningthe calibration data 1505. Thus, there is substantially zero percenterror in the data of FIG. 11 when P_(T) equals 20.6 s.

It is also noteworthy to observe that as P_(T) increases, the rate ofchange in the error decreases. Likewise, as P_(T) decreases, the rate ofchange of the error increases. One example of a situation that can giverise to small values of P_(T) is when the distance between the twoelectrodes in a conduction cell sensor decreases. For example, thedistance between the electrodes 1320 and 1325 in FIGS. 7A-C may bereduced by decreasing the thickness of the substantially nonconductivematerial 1310 and 1315. By decreasing this distance, the volume of thesample chamber 1340 also decreases. Thus, the influence of P_(T) on ananalyte estimate increases as the volume of the sample chamberdecreases, further highlighting the usefulness and importance of beingable to correct accurately for variations in P_(T) at small samplevolumes.

One example of using P_(T) to correct an analyte estimate for errorscaused by variations to P_(T) includes determining a relationshipbetween P_(T) and the time t needed for potentiometric relaxation toreach a particular potential difference. In this example, a DC potentialdifference of 400 mV was applied to the electrodes for the simulatedsystem of FIG. 3. Once a steady-state current was established, thepotential difference was removed by open-circuiting the system—therebyensuring that substantially no electronic current flowed in thecircuitry—and the potential between the electrodes was monitored overtime.

The simulation was executed for different values of P_(T), and therelaxation of the potential difference over time was determined for eachof these different values of P_(T). The evolution of this potential overtime has been modeled and is presented in FIG. 5 for various values ofP_(T). In this figure, the potential is determined as it is relaxingafter the circuit has been opened at time t=0 for the following valuesof P_(T)=28.7 s 420; P_(T)=19.2 s 415; P_(T)=11.6 s 410; P_(T)=5.9 s405; P_(T)=3.8 s 400.

The rate of relaxation of the potential between the two electrodes issubstantially independent of analyte concentration. Thus, the usefulnessof this technique is that it can be used to monitor properties of thesystem that give rise to the value of P_(T) while not beingsubstantially influenced by the analyte concentration. This is in starkcontrast to prior-art methods which rely on monitoring relaxationcurrents, which are substantially influenced by analyte concentration(U.S. patents: U.S. Pat. No. 5,942,102, U.S. Pat. No. 6,179,979, U.S.Pat. No. 6,284,125).

Furthermore, monitoring the potential difference between the electrodesis a measure that is substantially independent of the area of theelectrodes. This has the useful benefit of removing yet anotherpotential source of variation from the measurement. This contrasts withmethods that rely on monitoring relaxation currents, since the measuredcurrent value is dependent on the effective electrode area.

One example embodiment for extracting a measure of P_(T) is to determinethe time taken from the start of potentiometric relaxation until aparticular value of the potential is reached. This represents one metricfor quantifying a measure of the rate of relaxation—or the time constantfor the rate of decay—of the potential upon open circuiting theelectrochemical system. One of ordinary skill in the art will recognizethat other metrics can be used, such as the potential at a particularpoint in time, the slope of the potential vs. time plot during aparticular period of time, the slope of the plot of the logarithm ofpotential vs. time during a particular period of time, and the slope ofthe plot of 1/V² vs. time during a particular period of time, where V isthe potential. FIG. 12 illustrates a relationship between P_(T) and thetime t taken to reach a potential difference of 0.06 V, where t=0 is thetime at which the system is open-circuited. In this example, data shownin FIG. 6 was analyzed by determining the time at which the relaxingpotential reached a value substantially equal to 0.06 V. Thus, eachvalue of P_(T) results in a different decaying potential and correspondsto a different time at which the potential reached a value that issubstantially equal to 0.06V. FIG. 12, therefore, illustrates therelationship that exists between P_(T) and a measure of P_(T)—in thisexample, the time taken to reach 0.06V and so allows a mathematicalrelationship to be established to determine P_(T) from a measurablequantity of the potentiometric relaxation data. In this example, thefollowing relationship is observed between P_(T) and the time to reachthe designated potential:

P _(T) =mt

m=56.057

Other relationships may exist and such relationships may depend on themeasurement set up, including the geometry of the electrochemical cell.

Since this potentiometric relaxation measurement is substantiallyindependent of the analyte concentration, it can be used to estimate ameasure of P_(T) that is substantially unaffected by analyteconcentration. This measure of P_(T) may then be used to adjust forerrors in the estimate of analyte that may arise from variations in thevalue of P_(T). In this example, a calibration curve was determined fora system with P_(T)=20.6 s. As discussed earlier, one equation toestimate the concentration of an analyte may be given by:

$P_{T} = {- \frac{h}{s}}$$C = {{- \frac{K_{cell}I_{S}}{\beta_{S}}} = {{- \frac{I_{S}h}{\beta_{S}A}} = \frac{I_{S}P_{T}}{\beta \; A}}}$

in terms of the mobility of the analyte species. One of ordinary skillin the art will recognize that other forms are possible, depending onthe nature of the system. In one example discussed earlier, theconcentration of an analyte may be given in terms of its diffusionproperties by the following:

$P_{T} = \frac{h}{D}$$C = {\frac{\alpha \; K_{cell}I_{S}}{D} = {\frac{I_{S}h}{2{nFDA}} = {\frac{I_{S}P_{T}}{2{nFA}} = \frac{\alpha \; I_{S}P_{T}}{A}}}}$$\alpha = \frac{1}{2{nF}}$

In practice, a calibration curve is determined empirically since it isoften more convenient to determine a proportionality constant byexperimentation than determining all of the relevant componentsindependently. For example, taking the system described in terms ofanalyte mobility as discussed above, one equation to describe thecalibration data 1505 in FIG. 10 is:

$I = {{{\frac{1}{\lambda}\lbrack{ferrocyanide}\rbrack}\lbrack{ferrocyanide}\rbrack} = {\lambda \; I}}$$\lambda = {{2.14E\; 5{{mM}/{A\lbrack{ferrocyanide}\rbrack}}} = {{\lambda \; I} = \frac{{IP}_{T}}{\beta \; A_{e}}}}$$\lambda = \frac{P_{T}}{\beta \; A_{e}}$ A = 1  cm²$P_{T} = {\left. 20.6_{S}\Rightarrow\beta \right. = {\left. {{9.63E} - 5}\Rightarrow\lambda \right. = {\frac{P_{T}}{{9.63E} - 5} = {\left( {1.03E\; 4} \right)P_{T}}}}}$

The proportionality constant incorporates the effects of the relevantparameters on the concentration estimate. Since this data 1505 wasacquired on a system with a value of P_(T) equal to 20.6 s, the constantλ may be expressed to explicitly incorporate the value of P_(T), asindicated above. This then allows for a useful adjustment to be made forvariations in P_(T) that may introduce errors into the analyte estimate.

One example of correcting for errors in analyte concentration estimatesthat may arise from variations in P_(T) is to adjust the description ofthe calibration curve to account for variations in P_(T). Moregenerally, the proportionality constant of a calibration curve λ may begiven relative to the value of P_(T) that was associated with thecalibration data, indicated by P_(T calibration). In this example, thevalue of λ can then be adjusted by the quantity

$\left( \frac{P_{Tcalibration}}{P_{Tmeasured}} \right)$

such that:

$\lambda = {2.14 \times 10^{5}\left( \frac{P_{Tcalibration}}{P_{Tmeasured}} \right)}$

where P_(Tmeasured) is the value of P_(T) associated with the measuredcurrent signal I_(measured). This then allows for adjusting a parameterλ—that, in part, defines a calibration curve—in response to variationsin P_(T) when measuring a sample. Thus, by obtaining a measure of P_(T)that is substantially independent of I_(measured) and the analyteconcentration, the calibration curve that is used for estimating theanalyte concentration may be adjusted to reduce an error in estimate ofthe analyte concentration. Embodiments of the invention provide a methodfor determining P_(T) that is substantially independent of analyteconcentration and measured current. One embodiment of the method toadjust for a variation in P_(T) is to use embodiments of this inventionto determine a measure of P_(T) and adjust a parameter that in partdefines a calibration curve. One example embodiment of this method is tomultiply the calibration factor λ by the correction factor

$\left( \frac{P_{Tcalibration}}{P_{Tmeasured}} \right)$

and then use this adjusted value of λ in estimating the analyteconcentration. The following equations illustrate this exampleembodiment. From calibration data 1505 in FIG. 10, an equation isdetermined that substantially models this data. One example embodimentin determining such an equation is to use well-known linear regressiontechniques to find a linear equation that best describes this data 1505.One example of such an equation is given by the following:

C _(calibration)=(λ_(calibration))(I _(S) _(calibration) )

C _(estimated)=(λ_(calibration))(I _(S) _(measured) )

λ_(calibration)=(1.03E4)P _(T) _(calibration)

P _(Tcalibration)=20.6

λ_(calibration)=2.14×10⁵

Other relationships are possible and may depend on the nature of theelectrochemical sensor system, environmental factors, apparatus factors,and/or sample factors.

Another example of correcting for errors in analyte concentrationestimate that may arise from variations in P_(T) is to adjust themeasured current to account for variations in P_(T). A similar analysesof the calibration curve equation shows that the measured current I_(S)_(measured) may be adjusted by a factor of

$\left( \frac{P_{Tcalibration}}{P_{Tmeasured}} \right)$

as follows:

  C_(estimated) = (λ_(calibration))(I_(S_(measured)))$\mspace{20mu} {I_{S_{corrected}} = {I_{S_{measured}}\left( \frac{P_{Tcalibration}}{P_{Tmeasured}} \right)}}$$C_{corrected} = {{\left( \lambda_{calibration} \right)\left( I_{S_{corrected}} \right)} = {\left( \lambda_{calibration} \right){I_{S_{measured}}\left( \frac{P_{Tcalibration}}{P_{Tmeasured}} \right)}}}$

Another example of correcting for errors in analyte concentrationestimate that may arise from variations in P_(T) is to adjust theestimated analyte concentration to account for variations in P_(T). Asimilar analyses of the calibration curve equation shows that theestimated concentration C_(estimated) may be adjusted by a factor of

$\left( \frac{P_{Tcalibration}}{P_{Tmeasured}} \right)$

as follows:

  C_(estimated) = (λ_(calibration))(I_(S_(measured)))$C_{corrected} = {{\left( \lambda_{calibration} \right){I_{S_{measured}}\left( \frac{P_{Tcalibration}}{P_{Tmeasured}} \right)}} = {C_{estimated}\left( \frac{P_{Tcalibration}}{P_{Tmeasured}} \right)}}$

other adjustments may be made to correct for variations in P_(T) andthat the form of the adjustment depends on apparatus factors,environmental factors, and/or sample factors that are relevant to themeasurement system.

FIG. 10 illustrates a difference in calibration curves that can resultfrom different values of P_(T). Data points 1510 were from a system withP_(T)=6.58 s; data points 1505 were from a system with P_(T)=20.6; datapoints 1500 were from a system with P_(T)=44.3 s. It is clear that asthe value of P_(T) changes, sensor response changes, as reflected by thedifferent calibration curves. Thus, if a sensor system were developedand calibrated with a particular value of P_(T), the value of P_(T) whenthe sensor was used should be substantially the same in order tomaintain substantially the same sensor reading. However, if the value ofP_(T) were different when the sensor was used, then the measurement maybe inaccurate. Examples of why the value of P_(T) may be different atthe time of use of a sensor include manufacturing variations in thedistance between the electrodes of the conduction cell and variations inthe effective mobility of the species in the sample.

FIG. 11 illustrates an example of the type of error that can arise fromvariations in P_(T). In this example, the system of FIG. 2 implementedwith the processes of FIG. 3 is simulated with P_(T)=20.6 s. The errorin estimating analyte concentration is expressed as a percent of thevalue obtained with P_(T)=20.6 s. Thus, as P_(T) decreases below 20.6 s,the estimated analyte concentration increases at a rapid rate, resultingin a falsely high estimate; as P_(T) increases above 20.6 s, theestimated concentration decreases, resulting in a falsely low estimate.It is important to note that at small values of P_(T), the estimation ofanalyte concentration is much more sensitive to variations in P_(T) thanat larger values of P_(T). At small volumes, where the spacing betweenthe electrodes is small, the ability to correct for variations in P_(T)is thus of even greater value and usefulness. Likewise, in systemscomprising species with high mobilities and/or high diffusion, this alsocan result in small values of P_(T), again illustrating the usefulnessand value of the invention in correcting for variations in P_(T).

FIGS. 13A-C illustrates scenarios for correcting an estimate due tovariations in P_(T). In these examples, an analyte concentration of 3 mMis used with a system calibrated with P_(T)=20.6 s. In FIGS. 13A, 13B,and 13C, data points 1700 show how the estimated analyte concentrationcan vary when measurements are made with sensors with different valuesof P_(T). As expected, when the value of the sensor is substantially20.6 s, then the estimated concentration is substantially close to thecorrect value of 3 mM. FIG. 13A illustrates the effect of correcting thefinal estimated analyte concentration by accounting for variation inP_(T). Data points 1705 are the estimated analyte concentration after acorrection process was used that adjusts the estimated concentrationvalues in data points 1700 based on the value of P_(T) for that sensormeasurement. FIG. 13B illustrates the effect of correcting the finalestimated analyte concentration by adjusting the calibration curve toaccount for variation in P_(T). Data points 1710 are the estimatedanalyte concentration after a correction process was used that adjuststhe calibration curve that is used to estimate the concentration valuesin data points 1700 based on the value of P_(T) for that sensormeasurement. FIG. 13C illustrates the effect of correcting the finalestimated analyte concentration by adjusting the measured current signalto account for variation in P_(T). Data points 1715 are the estimatedanalyte concentration after a correction process was used that adjuststhe measured amperometric signal that is used in estimating theconcentration values in data points 1700 based on the value of P_(T) forthat sensor measurement. Thus, it is evident that embodiments of theinvention are useful in reducing errors in estimating analyteconcentration that can arise from variations in P_(T).

FIGS. 14A-C illustrate in flow-chart form example embodiments of theinvention. As discussed in this document, there are differentembodiments that can be used for the method of the invention. FIGS.14A-C give further detail to the steps of FIG. 4 by illustrating exampleembodiments of step 225, step 230, and step 235 of FIG. 4.

FIG. 14A illustrates one example embodiment in which an adjustment ismade to the final analyte estimate, as was illustrated by the example ofFIG. 13A. In this embodiment, the transient potential is determined(step 220). Then a measure of P_(T) is quantified (step 1805). Themeasure of P_(T) is compared to calibration data to determine aneffective value of P_(T) (step 1810). The effective value of P_(T) isused to adjust the analyte estimate to account for variations in P_(T)(step 1815). The adjusted analyte concentration is output in a usefulform (step 1820).

FIG. 14B illustrates one example embodiment in which an adjustment ismade to a measure of the Faradaic signal component, as was illustratedby the example of FIG. 13B. In this embodiment, the transient potentialis determined (step 220). Then a measure of P_(T) is quantified (step1805). The measure of P_(T) is compared to calibration data to determinean effective value of P_(T) (step 1810). The effective value of P_(T) isused to adjust the measure of the Faradaic signal component to accountfor variations in P_(T) (step 1825). The adjusted measure of theFaradaic signal component is used with the Faradaic calibration data toestimate the analyte concentration (step 1830). The estimate of theanalyte concentration is output in a useful form (step 1820).

FIG. 14C illustrates one example embodiment in which an adjustment ismade to calibration data, as was illustrated by the example of FIG. 13C.In this embodiment, the transient potential is determined (step 220).Then a measure of P_(T) is quantified (step 1805). The measure of P_(T)is compared to calibration data to determine an effective value of P_(T)(step 1810). The effective value of P_(T) is used to adjust Faradaiccalibration data to account for variations in P_(T) (step 1835). Theadjusted Faradaic calibration data is used with the measure of theFaradaic signal component to estimate the analyte concentration (step1840). The estimate of the analyte concentration is output in a usefulform (step 1820).

Example 2 Enzyme Biosensor Example

In another example embodiment, the conduction-cell electrochemicalsensor is operated as a biosensor. In this case, a set of chemicalreactions produces an analyte to be detected by the conduction-cellelectrochemical sensor. One embodiment of this is illustrated in FIG. 1where an enzyme glucose oxidase catalyzes a reaction with glucose. Inthis example, glucose 500 reacts with the oxidized form of glucoseoxidase, GODox 510, thereby converting the enzyme to its reduced formGODred 515 and producing gluconolactone 505. GODred 515 can react withferricyanide, Fe(CN)63—525 to be returned to its oxidized state GODox510 and produce ferrocyanide, Fe(CN)64—520. Thus, the concentration ofglucose may be estimated by determining the concentration offerrocyanide by the method and apparatus of embodiments of theinvention.

The measured current can be related to the glucose concentration by acalibration curve equation. One example of such an equation is:

C _(estimated) ^(glucose)=(λ_(calibration) ^(glucase))(I _(S)_(measured) )

where C_(estimated) ^(glucose) is the estimated glucose concentration,λ_(calibration) ^(glucose) is a proportionality constant, andI_(S measured) is the measured current. One example of a correctionfactor that can be used to correct for variations in P_(T) is to followthe process described above for a conduction-cell electrochemical sensorand develop an analogous correction equation whereby the measured P_(T)value is used to adjust the calibration curve, the measured current, orthe concentration estimate as follows:

  C_(estimated)^(glucose) = (λ_(calibration)^(glucose))(I_(S_(measured)))$C_{corrected}^{glucose} = {{\left( \lambda_{calibration}^{glucose} \right){I_{S_{measured}}\left( \frac{P_{Tcalibration}^{glucose}}{P_{Tmeasured}^{glucose}} \right)}} = {C_{estimated}^{glucose}\left( \frac{P_{Tcalibration}^{glucose}}{P_{Tmeasured}^{glucose}} \right)}}$

where P_(T calibration) ^(glucose) is the value of P_(T) obtained whendetermining a calibration curve for a conduction-cell biosensor used toestimate glucose concentrations, P_(T measured) ^(glucose) is the valueof P_(T) obtained when measuring glucose in a conduction-cell biosensor,and C_(corrected) ^(glucose) is the corrected glucose concentration.

Example 3 Example Embodiments of Transducer Control Apparatus

One example embodiment of the transducer control apparatus 5 in FIG. 2is discussed. FIG. 15 illustrates a schematic for one example embodimentof the Transducer control apparatus 5. Two electrodes 2100 and 2105 arecoupled to the TCS 5 by substantially conductive paths 2110 and 2115. Astimulus application unit 2150 can apply a potential difference betweensubstantially conductive paths 2110 and 2130. The stimulus applicationunit 2150 can vary the potential difference in time. A currentquantification unit 2120 monitors the current that flows along lead2110. One of ordinary skill in the art will recognize that the currentflowing along lead 2110 is substantially the same as the current flowingalong leads 2115 and 2130. The current quantification unit 2120 may beconnected to lead 2115 or 2130 instead of to lead 2110. This embodimentdoes not limit the invention. A switch unit 2135 allows the ability toelectrically connect or disconnect lead 2130 to lead 2115. This switchunit enables the TCA 5 to force the current flow to be substantiallyzero amps. One of ordinary skill will recognize that other methods arepossible to force current flow to be substantially zero amps, such asswitching to a high-impedance circuit element. Examples of a switch unit2135 include a solid-state switch such as a MOSFET switch (e.g. AD417chip from Analog Devices); an electro-mechanical switch; and amechanical switch.

The switch unit 2135 may be connected in a manner so as to enable theshort-circuiting and/or open-circuiting of lead 2110 instead. One ofordinary skill in the art will recognize that there are differentplacement and operational options for changing between one operatingmode where current flow is not substantially impeded to anotheroperating mode where current flow is substantially impeded. A potentialquantification unit 2125 monitors the potential difference between lead2115 and lead 2110. This potential difference is related to thepotential difference between electrode 2105 and 2100. In a preferredembodiment, the potential at lead 2115 is substantially equal to thepotential at electrode 2105 and the potential at lead 2110 issubstantially equal to the potential at electrode 2100.

When the TCA 5 is operating in amperometric mode, switch 2135 is in aclose-circuit operation mode, a potential difference is applied by thestimulus application unit 2150 and the resulting current is monitored bythe current quantification unit 2120 and the potential is monitored bythe potential quantification unit 2125. When the system switches to apotentiometric operation mode, switch 2135 changes to an open-circuit—orhigh impedance—operation mode, the current quantification unit 2120monitors the current flow—which is expected to be substantially zeroamps—and the potential quantification unit monitors the potentialdifference—which is expected to follow a relaxation decay over time.

A more specific example embodiment for the TCA 5 is illustrated in FIG.16.

Circuit element 2220 represents a substantially conductive path to areference potential, such as ground. All thick solid lines representsubstantially electrically conductive paths (“leads”). The stimuluspotential is applied between lead 2215 and ground lead 2220. Whenswitching unit 2230 is in a close-circuit mode, lead 2225 and lead 2250are at substantially the same potential and amplifier 2200 maintainssubstantially the same potential at lead 2250 as is present at lead2215. One example of such an amplifier 2200 is an operational amplifier(“op-amp”). Lead 2250 is connected to electrode 2240. A second amplifier2205 maintains one connection to ground lead 2220 and another connectionvia lead 2265 to electrode 2245. A feedback resistor 2255 connects lead2265 to lead 2260. The potential difference that exists between lead2260 and ground lead 2220 is related to the current that flows throughthe electrochemical cell. Another amplifier monitors the differencebetween lead 2250 and lead 2265, and thereby monitors substantially thesame potential that exists between electrode 2240 and electrode 2245.One example of such an amplifier 2210 is a differential amplifier.Another example is an instrumentation amplifier. The potentialdifference between lead 2270 and ground lead 2220 is related to thepotential difference between lead 2250 and lead 2265. When switchingunit 2230 is in open-circuit operation, the amplifier 2200 ensures thatsubstantially zero current flows along lead 2250 and electrode 2240.Thus, the potential at lead 2260 is substantially the same as thepotential at the ground lead 2220. the potential between the electrodes2240 and 2245 is monitored by amplifier 2210 and manifested by apotential difference between lead 2270 and ground lead 2220.

Example 4 Another Metric for Quantifying P_(T)

As mentioned above, another example embodiment for extracting a measureof P_(T) is to determine a measure of the rate of relaxation of thepotentiometric signal by determining a slope of 1/V² versus time duringa particular period of time, as illustrated by a schematic in FIG. 17.FIG. 17 illustrates a schematic representation of the potentiometricrelaxation signal as a function of time for different values of P_(T).In this example, the potentiometric relaxation was started at a time t=0by, for example, substantially open-circuiting the electrochemical cell.The y-axis plots the function of 1/V², where V is the measuredpotential, during a period of time from t1 until t2. Data traces 2400,2405, 2410, and 2415 represent the relaxation signals from measurementswith different values of P_(T). In this example, the value of P_(T)associated with data 2400 is smaller than the value of P_(T) associatedwith data 2405, which is in turn smaller than the value of P_(T)associated with data 2410, which is in turn smaller than the value ofP_(T) associated with data 2415. In this example, it is clear that thereis a substantially linear relationship between 1/V² and the time. Thus,a measure of the rate of relaxation may be obtained by determining aslope of this data in a period of time, for example between time t₁ andtime t₂. A slope may be calculated by well-established means in linearalgebra, including, but not limited to, a least squares method.

FIG. 18 illustrates in schematic form one example of quantifying ameasure of P_(T) from the measured data. A calibration curve can beconstructed that relates P_(T) to a measure of P_(T). The exampleillustrated by the schematic of FIG. 18 shows that some function of theslope of 1/V2 versus time—depicted as f(slope) on the y-axis—can be usedas a measure of P_(T). One example embodiment of such a function is:

${f({slope})} = \frac{1}{\sqrt{slope}}$

where slope is the slope of the plot of 1/V2 versus time during aportion of time. In FIG. 18, point 2500 corresponds to data trace 2400in FIG. 17; point 2505 corresponds to data trace 2405 in FIG. 17; point2510 corresponds to data trace 2410 in FIG. 17; point 2515 correspondsto data trace 2415 in FIG. 17. Thus, a measure of P_(T) can be computedfrom the measured potentiometric data. Once the measure of P_(T) isdetermined, this value can be used as discussed above to adjust variousquantities to obtain a more accurate estimate of analyte concentration.

Example 5 Performing Correction with Transient System

The invention may also be used in situations where the system has notreached steady state; such a state is also commonly known as a transientstate. Examples of a system in transient state include, but are notlimited to, a response signal whose characteristics vary with time, aconcentration profile in a sample which substantially varies with time,and a chemical reaction which has not reached equilibrium.

One difference between a steady-state system and a transient system isthat signals generated by a steady-state system are expected to containsignificant information about a distance term related to separationdistance between the electrodes and/or information about a transportterm related to the transport properties of the sample (for example, aneffective diffusion coefficient or a mobility term). For example, asdiscussed above P_(T) can be described in part by an effective electrodeseparation term h as well as other terms related to the transportproperties of the sample, such as a diffusion term D, a drift speed s,and/or a mobility term U. Signals generated by a transient system arenot expected to contain significant information about a distance termrelated to a separation distance between electrodes, but would beexpected to contain information related to the transport properties ofthe sample. However, even in transient systems, there may be significantinformation related to an effective distance term, though that distanceterm may not be directly related to a geometric separation distancebetween two electrodes.

In a steady state system, there is substantial transfer of chemicalinformation from one electrode to the other through the sample by theconcentration gradients of chemical species in the vicinity of oneelectrode being perturbed by the processes occurring at anotherelectrode. One example of this is when reaction products of oneelectrode reach the other electrode. In a transient system, there is notsubstantial chemical information transferred through the sample from oneelectrode to the other. For example, reaction products from oneelectrode may have not substantially reached the other electrode, so itis expected that there is little information in the signal about ameasure of the distance between the electrodes. There is, however,expected to be information about transport of analyte in the sample, forexample a transport term related to a diffusion term, a mobility term oran effective path length term. One example of such a path length term isan effective distance term related to transport of analyte in a samplecomprising substances such as red blood cells or other bodies. Bodies ina sample can affect the transport of analyte in a sample, and thereforean ability to correct for such transport-related variations in transientsystems would be useful and beneficial for increasing the accuracyand/or precision of a sensor system.

FIG. 19 illustrates in schematic form examples of amperometric signalsfor a transient system. In this example, three current traces are shownwhich correspond to different glucose concentrations. Trace 2900 is thelowest concentration, trace 2905 is the intermediate concentration andtrace 2910 is the highest concentration. The signals are divided intosix regions in time as shown. In this example, the sample was introducedinto the sample chamber at t=0, at which point the potential was steppedup to a level sufficient for a substantially diffusion limited currentto be generated. The increase in current from t=0 to t=t1 may beascribed predominantly to capacitative double layer charging. Thedecrease in current from t1 to t2 may be ascribed predominantly tostabilization of the double layer. The increase in current between t2and t3 may be ascribed predominantly to the increase of reducedmediator—ferrocyanide in this example—from the progress of the chemicalreaction with an analyte in the sample (in this example the chemicalreaction is the enzyme reaction with glucose). The current reaches alocal maximum at approximately time t3, at which point the progress ofthe enzyme reaction is balanced by the diffusion processed governingtransport of the electroactive species—the mediator in this example—tothe electrodes. resulting in a fall in current after time t3. Thefalling current in some cases continues to decrease is there is anapparent semi-infinite diffusion profile during the course of themeasurement.

Since the current signal continues to fall, it may be said that thissystem is in a transient state. The transient signal can be analyzed andquantified to determine a measure of the analyte concentration, and asteady state current is not necessary for the invention to be used. Oneexample of the type of analysis that can be performed with a transientcurrent is related to the Cottrell equation. Of course, the Cottrellequation applies to a particular set of measurement conditions and otherequations can be used to describe a system depending on the measurementconditions. One example equation that can be used is to compute asquare-root of a slope of 1/I² during a portion of time as a measure ofthe glucose concentration. In the example schematic of FIG. 19, aportion of time after the current substantially is independent of theenzyme reaction (for example, after a peak in the current atapproximately time t3) such as the time between t4 and t5 can be usedfor such quantification. In this example, an equation based on theCottrell equation can be used to describe the current of a transientsystem as follows:

$I = {{nFAC}\sqrt{\frac{D}{\pi \left( {t - t_{0}} \right)}}}$$\frac{1}{I^{2}} = {\left( \frac{\pi}{({nFA})^{2}} \right)\frac{1}{{DC}^{2}}\left( {t - t_{0}} \right)}$$\frac{1}{I^{2}} = {{\alpha \; t} + \beta}$$\alpha = {{\frac{\pi}{({nFA})^{2}{DC}^{2}}\beta} = {{- \left( \frac{\pi}{({nFA})^{2}} \right)}\frac{1}{{DC}^{2}}t_{0}}}$

where t_(o) is a reference time, and the other symbols retain theirusual meanings. The slope, given by α, and D can be used to determine ameasure of concentration, C. For a system that has a known value for D,it is therefore possible to quantify the concentration by determiningthe slope α, for example by an estimation equation such as:

$C = \sqrt{\frac{\pi}{({nFA})^{2}D\; \alpha}}$

A problem arises if sample, apparatus, and/or environmental factors varyand cause an unknown change in the apparent value of D. In such a case,the estimate of concentration, C, would subsequently vary in an unknownmanner, resulting in decreased accuracy and/or precision.

With such a transient system, the apparatus can be switched topotentiometric mode to monitor a potentiometric relaxation to determinea measure of the variations in transport and/or path length relatedproperties of the system. In this example, a potentiometric relaxationwould be used to determine an effective measure of D, which would beused to estimate the concentration. Thus, the method and apparatus ofthe invention does not require a steady-state or near-steady-statesystem; instead, the method and apparatus of the invention may be usedwith a transient system.

One factor that can influence the transient or steady-state nature ofthe system is the geometry of the electrochemical cell. One example ofsuch a geometric factor is the effective distance between theelectrodes. The smaller the effective distance, the less time itgenerally takes for the system to reach steady state. In this example,steady state can be defined as when the amperometric signal reaches anear steady-state value. The larger the effective distance between theelectrodes, the more time it generally takes to reach steady state.Thus, whether the system is in steady-state or transient mode when theoperation is switched from amperometric to potentiometric can depend onthe both the effective distance between the electrodes and the time atwhich the mode of operation changes. For example, if the effectiveseparation is large enough that steady state is not substantiallyachieved within a given period of time, then the mode of operation maybe switched in to potentiometric mode while in the transient state. Oneuseful benefit of using the method and apparatus of the invention with atransient system is that a measurement may be made in less time. As inthe steady state measurement in Example 4, for transient measurementsthere is a substantially linear relationship between 1/V² and the time.Thus, a measure of the rate of relaxation may be obtained by determininga slope of this data in a period of time, for example between time t₁and time t₂. A slope may be calculated by well-established means inlinear algebra, including, but not limited to, a least squares method.Other relationships may exist for different electrode geometries.

FIGS. 20A-C illustrate in schematic form several example amperometricsignals that may be generated by conduction-cell based biosensorsystems. FIG. 20A illustrates an example scenario where the effectiveseparation distance between the electrodes is large enough to allow atleast one portion of the response signal to follow a transient form thatmay be described by a relaxation related to the Cottrell equation. Thisfigure is similar to the example illustrated in FIG. 19 except that thesignal is shown to deviate from a substantially semi-infinite relaxationthat may be substantially related to a Cottrell-type relaxation (betweent4 and t5), go through a transition region (between t5 and t6) andultimately reach a substantial steady-state value (after t6). Thus,various equations and/or expressions can be used to describe the signalresponse during these different regions. FIG. 20B illustrates an examplescenario where the effective separation distance between the electrodesis large enough to allow a diffusion gradient relaxation (between t3 andt4) but small enough to reach a substantially steady-state current value(after t4) without significantly experiencing a substantiallysemi-infinite (e.g. Cottrell-like) relaxation. FIG. 20C illustrates anexample scenario where the effective separation distance between theelectrodes is small enough such that a substantially steady-statecurrent is reached (after t4) without experiencing a significantdiffusion relaxation. These figures are example embodiments of signalsthat may be generated by enzyme-based biosensors and do not limit theinvention. One of ordinary skill in the art will recognize that othersignal responses are possible and that the form of the signal depends onmany factors, including but not limited to apparatus factors, samplefactors, and environmental factors.

Another geometric factor that can influence the transient orsteady-state nature of the system is the orientation of the electrodes.A traditional orientation for conduction cells has been for cellscomprised of two substantially parallel electrodes facing each other,usually of approximately the same area, as illustrated by the example inFIGS. 7A-C. The principal reason for such a geometry has been that thisis a convenient electrode orientation for determining the cell constantK_(cell). However, the method and apparatus of the invention allowsother geometries to be used in a conduction-cell operation. Examplesinclude cells comprised of substantially coplanar electrodes and/orsubstantially concentric electrodes (examples of which are shown inFIGS. 21A-D).

FIGS. 21A-D illustrate in schematic form several example arrangements ofsubstantially coplanar electrodes. In these example illustrations, thearrangements are comprised of a substantially non-conducting substrate2000, at least two electrodes 2005 and 2020, and at least twosubstantially conducting leads 2010 and 2015. The substantiallyconducting leads (2010 and 2015) need not be separate material from theelectrodes (2005 and 2020). These illustrations are provided as exampleembodiments and do not limit the invention. One of ordinary skill in theart will recognize that other embodiments are possible, including butnot limited to different shapes, different orientations, and/ordifferent alignments of electrodes.

Other factors that can influence the transient or steady state nature ofthe system are sample properties, for example, diffusion, mobility, pathlength, and/or hematocrit level. Properties of the sample itself canalter the rate of transport of species, thereby altering the time neededto achieve substantially a steady-state system.

The invention therefore provides a method and apparatus for determininga measure of PT that does not require the system to be substantially insteady-state, but can be implemented on a transient system. This has theuseful benefit of substantially reducing the measurement time, since auser does not need to wait until substantial steady state has beenachieved. Another useful benefit is that different cell geometries canbe used, including, for example, parallel-facing, coplanar, and/orconcentric arrangements of electrodes, since such geometries may notquickly and easily achieve substantial steady-state. Furthermore, sincethe invention can be used with transient systems as well, there is norequirement that the electrodes must be sufficiently close to each otherto achieve a substantial steady-state operation. Electrochemical cellsmanufactured at these larger scales are known to be easier, and henceless costly, to make.

It will be apparent to those skilled in the art that additional variousmodifications and variations can be made without departing from thescope or spirit of the invention.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein.

REFERENCES CITED

-   Schmidt-Weinmar, von H. G. “Ueber die Polarisation einer    symmetrischen Redoxzelle mit kleinem Elektrodenabstand: Eine Methode    zur Bestimmung der Ionenbeweglichkeit”. Berichte der    Bunsengesellschaft. Vol 71, No. 1. 1967.-   Brett, C M A and A. M. O. Brett. “Electrochemistry: Principles,    Methods, and Applications”, 1st ed. Oxford University Press, 1993.-   MacInnes, D. A. “The Principles of Electrochemistry”. Reinhold    Publishing Corp. New York. 1939.-   Crow, D. R. “Principles and Applications of Electrochemistry”. 4th    ed. Stanley Thornes Publishers. Cheltenham, UK. 1998.-   Atkins, P. “Physical Chemistry”. 6th ed. Freeman. New York. 1999.-   Lathi, B. P. “Linear Systems and Signals”, Berkeley-Cambridge Press,    Carmichael, Calif. 1992.

1-22. (canceled)
 23. A method for increasing accuracy of a determinationof an analyte in a sample, comprising the steps of: (a) applying thesample to an electrochemical test strip; (b) applying a potential signalto the sample in the electrochemical test strip to result in a currentsignal that is indicative of the amount of analyte in the sample; (c)ceasing application of the potential signal and determining a transientpotential; (d) determining from the transient potential a correctionfactor for the effective transport parameter (P_(T)) or the electrodearea (A) or both the effective transport parameter (P_(T)) or theelectrode area (A), and (e) applying the correction factor(s) determinedin (d) to arrive at a final determination of the analyte in the sample,said final determination of the analyte in the sample being moreaccurate than an estimated analyte value derivable from the currentsignal without application of correction factor(s).
 24. The method ofclaim 23, wherein a measure of P_(T) is determined and compared to acalibration value of P_(T), and wherein an estimated analyte value isdetermined from the current signal, and then is adjusted to account fordifferences between the measured P_(T) and the calibration value ofP_(T) to arrive at the final determination of analyte.
 25. The method ofclaim 23, wherein a measure of P_(T) is determined and compared to acalibration value of P_(T), and the current signal is adjusted toaccount for differences between the measured P_(T) and the calibrationvalue of P_(T), and the final determination of analyte is calculatedfrom the adjusted current signal.
 26. The method of claim 23, wherein ameasure of P_(T) is determined and compared to a calibration value ofP_(T), and Faradaic calibration data is adjusted to the account fordifferences between the measured P_(T) and the calibration value ofP_(T), the adjusted Faradaic calibration data is applied to the currentsignal to produce a calibrated current signal, and a final determinationof analyte is calculated from the calibrated current signal.
 27. Themethod of claim 23, wherein the electrochemical test strip comprisesreagents for determination of glucose in a sample.
 28. The method ofclaim 27, wherein a measure of P_(T) is determined and compared to acalibration value of P_(T), and wherein an estimated analyte value isdetermined from the current signal, and then is adjusted to account fordifferences between the measured P_(T) and the calibration value ofP_(T) to arrive at the final determination of analyte.
 29. The method ofclaim 27, wherein a measure of P_(T) is determined and compared to acalibration value of P_(T), and the current signal is adjusted toaccount for differences between the measured P_(T) and the calibrationvalue of P_(T), and the final determination of analyte is calculatedfrom the adjusted current signal.
 30. The method of claim 27, wherein ameasure of P_(T) is determined and compared to a calibration value ofP_(T), and Faradaic calibration data is adjusted to the account fordifferences between the measured P_(T) and the calibration value ofP_(T), the adjusted Faradaic calibration data is applied to the currentsignal to produce a calibrated current signal, and a final determinationof analyte is calculated from the calibrated current signal.
 31. Themethod of claim 23, wherein the electrochemical test strip comprisesfacing working and counter electrodes.
 32. The method of claim 31,wherein a measure of P_(T) is determined and compared to a calibrationvalue of P_(T), and wherein an estimated analyte value is determinedfrom the current signal, and then is adjusted to account for differencesbetween the measured P_(T) and the calibration value of P_(T) to arriveat the final determination of analyte.
 33. The method of claim 31,wherein a measure of P_(T) is determined and compared to a calibrationvalue of P_(T), and the current signal is adjusted to account fordifferences between the measured P_(T) and the calibration value ofP_(T), and the final determination of analyte is calculated from theadjusted current signal.
 34. The method of claim 32, wherein a measureof P_(T) is determined and compared to a calibration value of P_(T), andFaradaic calibration data is adjusted to the account for differencesbetween the measured P_(T) and the calibration value of P_(T), theadjusted Faradaic calibration data is applied to the current signal toproduce a calibrated current signal, and a final determination ofanalyte is calculated from the calibrated current signal.
 35. The methodof claim 23, wherein the electrochemical test strip comprisesside-by-side electrodes.
 36. The method of claim 35, wherein a measureof P_(T) is determined and compared to a calibration value of P_(T), andwherein an estimated analyte value is determined from the currentsignal, and then is adjusted to account for differences between themeasured P_(T) and the calibration value of P_(T) to arrive at the finaldetermination of analyte.
 37. The method of claim 35, wherein a measureof P_(T) is determined and compared to a calibration value of P_(T), andthe current signal is adjusted to account for differences between themeasured P_(T) and the calibration value of P_(T), and the finaldetermination of analyte is calculated from the adjusted current signal.38. The method of claim 35, wherein a measure of P_(T) is determined andcompared to a calibration value of P_(T), and Faradaic calibration datais adjusted to the account for differences between the measured P_(T)and the calibration value of P_(T), the adjusted Faradaic calibrationdata is applied to the current signal to produce a calibrated currentsignal, and a final determination of analyte is calculated from thecalibrated current signal.
 39. An apparatus for determining an analytein a sample with increased accuracy when the apparatus is operativelyconnected to an electrochemical test strip specific for the analyte,said apparatus comprising: (a) circuitry for applying a potential signalto the sample in the electrochemical test strip to result in a currentsignal that is indicative of the amount of analyte in the sample; (b)circuitry for ceasing application of the potential signal anddetermining a transient potential; (c) circuitry for determining fromthe transient potential a correction factor for the effective transportparameter (P_(T)) or the electrode area (A) or both the effectivetransport parameter (P_(T)) or the electrode area (A), and (d) circuitryfor applying the correction factor(s) determined in (c) to arrive at afinal determination of the analyte in the sample, said finaldetermination of the analyte in the sample being more accurate than anestimated analyte value derivable from the current signal withoutapplication of correction factor(s).
 40. The apparatus of claim 39,wherein the circuitry of (c) determines a measure of P_(T) and comparesthis measure to a calibration value of P_(T), wherein the apparatusfurther comprises circuitry for determining an estimated analyte valuefrom the current signal, and wherein the circuitry of (d) adjusts theestimated analyte value to account for differences between the measuredP_(T) and the calibration value of P_(T) to arrive at the finaldetermination of analyte.
 41. The apparatus of claim 39, wherein ameasure of P_(T) is determined and compares this value to a calibrationvalue of P_(T), and circuitry of step (d) adjusts the current signal toaccount for differences between the measured P_(T) and the calibrationvalue of P_(T), and wherein the apparatus further comprises circuitryfor making the final determination of analyte from the adjusted currentsignal.
 42. The apparatus of claim 35, wherein the circuitry of (c)determines a measure of P_(T) and compares it to a calibration value ofP_(T), the apparatus further comprises data storage for storing Faradaiccalibration data; the circuitry of (d) adjusts the stored Faradaiccalibration to the account for differences between the measured P_(T)and the calibration value of P_(T), and the apparatus further comprisescircuitry for applying the adjusted Faradaic calibration data to thecurrent signal to produce a calibrated current signal, and for making afinal determination of analyte from the calibrated current signal.